Methods and compositions for increasing the anaerobic working capacity in tissues

ABSTRACT

Provided are compositions comprising beta-alanylhistidine peptides and/or beta-alanines, and methods for administering these peptides and amino acids. In one aspect, the compositions and methods cause an increase in the blood plasma concentrations of beta-alanine and/or creatine.

RELATED APPLICATIONS

This application is a continuation of allowed U.S. patent applicationSer. No. 12/231,240, filed Aug. 29, 2008, which is a continuation ofU.S. patent application Ser. No. 10/717,217, filed Nov. 18, 2003, nowU.S. Pat. No. 7,504,376, which claims the benefit of priority under 35U.S.C. § 119(e) of U.S. Provisional Application No. 60/462,238 filedApr. 10, 2003 and that is a continuation-in-part (CIP) of U.S.application Ser. No. 10/209,169, filed Jul. 30, 2002, now U.S. Pat. No.6,680,294, which is a continuation of U.S. application Ser. No.09/757,782, filed Jan. 9, 2001, now U.S. Pat. No. 6,426,361, which is acontinuation of U.S. application Ser. No. 09/318,530, filed May 25,1999, now U.S. Pat. No. 6,172,098, which is a divisional of U.S.application Ser. No. 08/909,513, filed Aug. 12, 1997, now U.S. Pat. No.5,965,596, which claims the benefit of foreign priority under 35 U.S.C.§ 119 to United Kingdom Application Nos. 9621914.2, filed Oct. 21, 1996,and 9616910.7, filed Aug. 12, 1996. The aforementioned applications areincorporated by reference in their entirety.

TECHNICAL FIELD

This invention relates to the fields of pharmaceuticals and physiology.In one aspect, the invention provides methods for increasing thebuffering capacity of muscles and decreasing muscle fatigue. Theinvention also provides methods and compositions for increasing theanaerobic working capacity of muscle and other tissues.

BACKGROUND

Natural food supplements are typically designed to compensate forreduced levels of nutrients in the modern human and animal diet. Inparticular, useful supplements increase the function of tissues whenconsumed. It can be particularly important to supplement the diets ofparticular classes of animals whose normal diet may be deficient innutrients available only from meat and animal products (e.g., humanvegetarians and other animals who consume an herbivorous diet).

For example, in the sporting and athletic community, natural foodsupplements which specifically improve athletic ability are increasinglyimportant, such as supplements that promote or enhance physical prowessfor leisure or employment purposes. In another example, anaerobic (e.g.,lactate-producing) stress can cause the onset of fatigue and discomfortthat can be experienced with intense exercise (e.g., continuous orintermittent sprinting in soccer or ice-hockey), where oxygenavailability may be limited (e.g., peripheral vascular disease, freediving or synchronized swimming) and with aging. Anaerobic stress canalso result from prolonged submaximal isometric exercise when the localcirculation is partially or totally occluded by the increase inintra-muscular pressure (e.g., during rock climbing). Excessive lactateproduction can result in the acidification of the intracellularenvironment.

Creatine (i.e., N-(aminoiminomethyl)-N-glycine, N-amidinosarcosine,N-methyl-N-guanylglycine, or methylglycocyamine) is found in largeamounts in skeletal muscle and other “excitable” tissues (e.g., smoothmuscle, cardiac muscle, or spermatozoa) characterized by a capacity forhigh and variable energy demand. Creatine is converted intophosphorylcreatine in energy-generating biochemical pathways withincells. In mammalian skeletal muscle, the typical combined content ofcreatine (i.e., creatine and phosphorylcreatine) may vary from less than25 to about 50 mmol per kilogram fresh muscle (i.e., 3.2 to 6.5 gramsper kilogram fresh muscle).

Creatine is formed in the liver and taken up into tissues, such asmuscle, by means of an active transport system. Creatine synthesis inthe body may also be augmented by the ingestion of creatine present inmeat (e.g., 5-10 milligrams per kilogram body weight per day in theaverage meat-eating human and approximately zero in a vegetarian diet).

During sustained intense exercise, or exercise sustained underconditions of local hypoxia, the accumulation of hydronium ions formedduring glycolysis and the accumulation of lactate (anaerobic metabolism)can severely reduce the intracellular pH. The reduced pH can compromisethe function of the creatine-phosphorylcreatine system. The decline inintracellular pH can affect other functions within the cells, such asthe function of the contractile proteins in muscle fibers.

Dipeptides (also referred to herein as peptides) of beta-alanine andhistidine, and their methylated analogues, which include carnosine(beta-alanyl-L-histidine), anserine (beta-alanyl-L-1-methylhistidine),or balenine (beta-alanyl-L-3-methylhistidine), are present in themuscles of humans and other vertebrates. Carnosine is found inappreciable amounts in muscles of, for example, humans and equines.Anserine and carnosine are found in muscles of, for example, canines,camelids and numerous avian species. Anserine is the predominantbeta-alanylhistidine dipeptide in many fish. Balenine is the predominantbeta-alanylhistidine dipeptide in some species of aquatic mammals andreptiles. In humans, equines, and camelids, the highest concentrationsof the beta-alanylhistidine dipeptides are found in fast-contractingglycolytic muscle fibers (type IIA and IIB) which are used extensivelyduring intense exercise. Lower concentrations are found in oxidativeslow-contracting muscle fibers (type I). See, e.g., Dunnett, M. &Harris, R. C. Equine Vet. J., Suppl. 18, 214-217 (1995). It is knownthat carnosine contributes to hydronium ion buffering capacity indifferent muscle fiber types, and up to 50% of the total in equine typeII fibers.

SUMMARY

The invention provides methods of increasing anaerobic working capacityin a tissue, comprising the following steps: (a) providing abeta-alanylhistidine dipeptide and a glycine, an insulin, an insulinmimic, or an insulin-action modifier; and (b) administering thebeta-alanine and at least one of the glycine, insulin mimic, orinsulin-action modifier to the tissue in an amount effective to increasebeta-alanylhistidine dipeptide synthesis in the tissue, therebyincreasing the anaerobic working capacity in the tissue. The inventionprovides methods of regulating hydronium ion concentrations in a tissuecomprising the following steps: (a) providing a beta-alanylhistidinedipeptide and a glycine, an insulin an insulin mimic, or aninsulin-action modifier; and (b) administering the beta-alanine and atleast one of the glycine, insulin mimic, or insulin-action modifier tothe tissue in an amount effective to increase the hydronium ionconcentration in the tissue.

In one aspect of the methods, the step of administering the beta-alanineand at least one of the glycine, insulin mimic, or insulin-actionmodifier to the tissue comprises oral administration, administration toa blood or blood plasma or a combination thereof. Thebeta-alanylhistidine dipeptide can comprise a carnosine, an anserine, ora balenine, or analogs or mimetics thereof.

The invention provides compositions comprising a mixture of a glycine,an insulin, an insulin mimic or an insulin-action modifier, and acomposition comprising an amino acid or an active derivative thereofselected from the group consisting of a beta-alanine, a chemicalderivative of beta-alanine and a peptide comprising a beta-alanine oranalogs thereof. In one aspect, the beta-alanine comprises abeta-alanylhistidine dipeptide, such as a carnosine, an anserine or abalenine or analogs thereof. The compositions can further comprise atleast a creatine or a carbohydrate.

In one aspect, the insulin mimic comprises a D-pinitol(3-O-methyl-chiroinositol), a 4-hydroxy isoleucine, ademethyl-asterriquinone B-1 compound, an alpha lipoic acid, an R-alphalipoic acid, a guanidiniopropionic acid, a vanadium compound, a vanadiumcomplex or a synthetic phosphoinositolglycan peptide. The insulin-actionmodifier can be a sulphonylurea, a thiazolidinedione or a biguanide.

In alternative aspects, the composition is a pharmaceutical composition,a dietary supplement or a sports drink. The dietary supplement or sportsdrink can be a supplement for humans. The pharmaceutical composition canbe formulated for humans.

The invention provides compositions comprising at least 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 or more grams of a peptide or anester comprising a beta-alanine or analogs or mimetics thereof. Theinvention provides compositions comprising at least about 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 or more gramsof a peptide or an ester comprising a beta-alanine (or analogs ormimetics thereof) in an injectable form. In one aspect, the peptidecomprises a beta-alanylhistidine dipeptide, such as a carnosine, ananserine or a balenine, or analogs or mimetics thereof.

The invention provides compositions formulated for humans comprising atleast 200, 225, 250, 275, 300, 325, 350, 400, 425, 450, 475, 500, 525,550, 575, 600, 625, 650, 675, 700, 750, 775, 800, 825, 850, 875, 900,925, 950, 975 or 1000 or more mg of a beta-alanine or beta-alanineanalogs or mimetics. In one aspect, the composition is formulated in aningestible or an injectable formulation. The ingestible formulation canbe a drink, a gel, a food or a tablet. The peptide can comprise abeta-alanylhistidine dipeptide, such as a carnosine, an anserine or abalenine, or analogs or mimetics thereof.

The invention provides methods of increasing the anaerobic workingcapacity of a tissue in a subject comprising the following steps: (a)providing a composition comprising (i) a mixture of a glycine, aninsulin, an insulin mimic or an insulin-action modifier, and acomposition comprising an amino acid or an active derivative thereofselected from the group consisting of a beta-alanine, a chemicalderivative of beta-alanine and a peptide comprising a beta-alanine, oranalogs or mimetics thereof; (ii) at least 0.5 gram of a peptide or anester comprising a beta-alanine in an injectable form; or, (iii) atleast 200 mg of a beta-alanine; and (b) administering the composition tothe subject in an amount effective to increase the anaerobic workingcapacity of the tissue. In one aspect, the total dosage of thebeta-alanine for a 24-hour period is at least about 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5, 3.0,3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 or more grams. Thetotal dosage of the beta-alanine for a 24-hour period can be betweenabout 0.2 gram and about 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 or moregrams. The composition can be given over a period of at least 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more days. The composition canbe given over a period of at least about 3 days to about two, three,four or more weeks. The beta-alanine can comprise a beta-alanylhistidinedipeptide, such as a carnosine, an anserine or a balenine, or analogs ormimetics thereof. The total dosage of the beta-alanylhistidine dipeptideover a 24 hour period can be at least about 0.5 gram, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,5.5, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 or more grams. The total dosage of thebeta-alanylhistidine dipeptide over a 24 hour period can be greater thanabout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 ormore grams. The total dosage of the beta-alanylhistidine dipeptide overa 24 hour period can be more than about 5 gram to about 16 gram. Thecomposition can be administered in multiple doses. The composition canbe administered at least two times to eight times in a 24-hour period.In one aspect, about 200 mg, 225, 250, 275, 300, 325, 350, 400, 425,450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 750, 775, 800,825, 850, 875, 900, 925, 950, 975 or 1000 mg of a beta-alanine (oranalogs or mimetics thereof) and/or about 500 mg (or, about 200 mg, 225,250, 275, 300, 325, 350, 400, 425, 450, 475, 500, 525, 550, 575, 600,625, 650, 675, 700, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975 or1000 mg) of carnosine (or analogs or mimetics thereof) is administeredabout two to eight, or more, times a day (e.g., 2, 3, 4, 5, 6, 7, 8 ormore times a day) over a period of several weeks. In one aspect, atleast about 2 g of a beta-alanine or at least about 5 g of carnosine isadministered about two to eight times a day over a period of about two,three or four days.

In one aspect, the amount of a composition of the invention administeredis increased daily. The amount of the composition of the inventionadministered can be increased weekly. The composition can beadministered in treatment periods that last for at least about fourweeks.

While the invention is not limited by any particular mechanism ofaction, the invention provides methods of regulating hydronium ionconcentration in tissue in a subject comprising the following steps: (a)providing a composition comprising (i) a mixture of a glycine, aninsulin, an insulin mimic or an insulin-action modifier, and acomposition comprising an amino acid or an active derivative thereofselected from the group consisting of a beta-alanine, a chemicalderivative of beta-alanine and a peptide comprising a beta-alanine oranalogs or mimetics thereof; (ii) at least 0.5 gram of a peptide or anester comprising a beta-alanine in an injectable form; or, (iii) atleast 200 mg of a beta-alanine; and (b) administering the composition tothe subject in an amount effective to regulate the hydronium ionconcentration in the tissue.

In one aspect, the invention features methods and compositions forincreasing the anaerobic working capacity of muscle and other tissues.The methods and compositions of the invention provide for thesimultaneous accumulation of creatine and/or beta-alanylhistidinedipeptides, or beta-alanine and L-histidine analogues, within a tissuein the body. The methods include ingesting or infusing compositions intothe body. In one aspect, the compositions are mixtures of compoundscapable of increasing the availability and uptake of creatine and ofprecursors for the synthesis and accumulation of beta-alanylhistidinedipeptides in human and animal tissue. The compositions of the inventioncan induce the synthesis and accumulation of beta-alanylhistidinedipeptides in a human or animal body when introduced into the body.

The compositions can include beta-alanine, chemical derivatives andanalogs of beta-alanine such as esters of beta-alanine, peptides ofbeta-alanine, such as carnosine, anserine, and balenine, as well asanalogues thereof. The compositions may also include L-histidine andmixtures thereof. Each of the beta-alanine and/or L-histidine can beformulated or administered as individual amino acids, or, as componentsof dipeptides (e.g., carnosine, anserine, and/or balenine),oligopeptides, or polypeptides. The beta-alanine, L-histidine,carnosine, anserine, and/or balenine, or peptides of beta-alanine can beactive derivatives. An active derivative is a compound derived from, oris a precursor of, a substance and performs in the same or similar wayin the body as the substance, or which is processed into the substancewhen placed into the body. Examples include, for example, esters andamides. Compositions can also include any one or more of a creatine, acarbohydrate, insulin, an insulin mimic, an insulin-action modifier or aglycine. The compositions of the invention can be used for thepreparation of a dietary supplement (including, e.g., drinks, gels,foods) or pharmaceutical composition for humans or animals. Thecompositions of the invention can be used in any of the methods of theinvention.

In one aspect, the invention features compositions for and a method ofregulating hydronium ion concentrations in a tissue. The method includesthe steps of providing an amount of beta-alanine to blood or bloodplasma effective to increase beta-alanylhistidine dipeptide synthesis ina tissue and exposing the tissue to the blood or blood plasma, wherebythe concentration of beta-alanylhistidine is increased in the tissue.The beta-alanylhistidine may be a carnosine, anserine, or a balenine.The method can include the step of providing an amount of L-histidine tothe blood or blood plasma effective to increase beta-alanylhistidinedipeptide synthesis.

In another aspect, the invention features a method of increasing theanaerobic working capacity of a tissue. The method includes the steps ofproviding an amount of beta-alanine to blood or blood plasma effectiveto increase beta-alanylhistidine dipeptide synthesis in a tissue,providing an amount of L-histidine to the blood or blood plasmaeffective to increase beta-alanylhistidine dipeptide synthesis in atissue, and exposing the tissue to the blood or blood plasma. Theconcentration of beta-alanylhistidine is increased in the tissue.

In alternative aspects, the methods can include the step of increasing aconcentration of creatine in the tissue. The increasing step can includeproviding an amount of creatine to the blood or blood plasma effectiveto increase the concentration of creatine in the tissue (e.g., byproviding creatine to the blood or blood plasma).

The providing steps of the methods can include ingestion, infusion(e.g., injection) or a combination of ingestion and infusion, of acomposition including an amount of beta-alanine, a peptide ofbeta-alanine such as carnosine, anserine and balenine which arehydrolyzed to their constituent amino acids on ingestion and are asource of beta-alanine for the body. Methods of the invention alsoinclude providing L-histidine, creatine, carbohydrate, insulin, insulinmimics, insulin-action modifiers and/or glycine.

In yet another aspect, the methods can include increasing aconcentration of insulin in the blood or blood plasma. The concentrationof insulin can be increased, for example, by injection of insulin.Methods of the invention can also include injection ingestion, or othermodes of delivery, known to those of skill in the art, to a body (alsoreferred to as a subject) of insulin mimics. Examples of insulin mimicsinclude, but are not limited to, D-pinitol (3-O-methyl-chiroinositol),4-hydroxy isoleucine, L783,281 (a demethyl-asterriquinone B-1 compound),alpha lipoic acid, R-alpha lipoic acid, guanidiniopropionic acid,vanadium compounds such as vanadyl sulfate or vanadium complexes such asperoxovanadium, and synthetic phosphoinositolglycans (PIG peptides).Additionally or alternatively, methods of the invention can include theuse of insulin-action modifiers to enhance or inhibit the action ofinsulin in the body. Examples of insulin-action modifiers can include,but are not limited to, sulphonylureas, thiazolidinediones, andbiguanides.

In still another aspect, the methods include providing glycine to abody. It is thought that glycine may suppress blood glucose release inthe blood after ingestion of a meal. It may be that glycine enhancesinsulin sensitivity by promoting greater glucose uptake. Accordingly,the methods include providing glycine alone or in conjunction withinsulin, insulin mimics or insulin-action modifiers in the compositionsand methods of the invention. Glycine may be provided in various forms,for example, alone or in combination with other substances, such as indietary supplements. Alternatively, glycine can be derived from othersources, such as gelatin.

The tissue referred to in the invention can be a skeletal muscle.

In one aspect, the invention provides compositions for practicing themethods of the invention. Accordingly, one aspect of the inventioncontemplates a composition having one or more active ingredient,including beta-alanine, beta-alanylhistidine peptides (or analogues orderivatives thereof), creatine, insulin, insulin mimics orinsulin-action modifiers, glycine, and carbohydrate, to carry out themethods of the invention. The invention further contemplates the use ofmultiple compositions formulated to provide one or more activeingredient to the body for carrying out the methods of the invention.

Therefore, in an exemplary aspect, the invention features a compositionconsisting essentially of beta-alanine or a peptide source ofbeta-alanine, between about 39 and about 99 percent by weight of acarbohydrate, and up to about 60 percent by weight of water. Thecomposition can include between about 0.1 and about 20 percent by weightof the beta-alanine (in the free or a bound form). The composition caninclude between about 0.1 and about 20 percent by weight of L-histidine.

The carbohydrate can be a simple carbohydrate (e.g., glucose).

In another aspect, the invention features a composition consistingessentially of beta-alanine or a peptide source of beta-alanine, betweenabout 1 and about 98 percent by weight of a creatine source, and up toabout 97 percent by weight of water. The composition includes betweenabout 0.1 and about 98 percent by weight of the beta-alanine. Thepeptide source can include L-histidine and the composition can includebetween about 0.1 and about 98 percent by weight of L-histidine fromthis source.

The peptide source can be a mixture of amino acids, dipeptides,oligopeptides, polypeptides, or active derivatives thereof.

The composition can be a dietary supplement. The creatine source can becreatine monohydrate.

The concentrations of components in blood or blood plasma, includingbeta-alanine, can be increased by infusion (i.e., injection) oringestion of an agent operable to cause an increase in the blood plasmaconcentration. The composition can be ingested in doses of between about100 milligrams and about 800 grams or more per day. The doses can beadministered in one part or multiple parts each day.

An increase of creatine and beta-alanylhistidine dipeptides in themuscles can increase the tolerance of the cells to an increase inhydronium ion production with anaerobic work and lead to an increase inendurance during exercise before the onset of fatigue. The compositionsand methods can contribute to correcting the loss of beta-alanine,L-histidine, or creatine due to degradation or leaching of theseconstituents during the cooking or processing of food. The compositionsand methods can also contribute to correcting the absence of thesecomponents from a vegetarian diet.

The methods and compositions can be used to increasebeta-alanylhistidine dipeptides in sportsmen, athletes, body-builders,synchronized swimmers, soldiers, elderly people, horses in competition,working and racing dogs, and game birds, to avoid or delay the onset ofmuscular fatigue.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other advantagesand features of the invention will be apparent to the skilled artisanfrom the detailed description, drawings, and claims.

All publications, patents, patent applications cited herein are herebyexpressly incorporated by reference for all purposes.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph depicting changes in the concentrations ofbeta-alanine in blood plasma of five horses, before and at 2 hourintervals following the feeding of beta-alanine and L-histidine (100milligrams per kilogram body weight and 12.5 milligrams per kilogrambody weight, respectively, three times per day) over a period of 30days.

FIG. 2 is a graph depicting changes in the concentrations of L-histidinein blood plasma of five horses, before and at 2 hour intervals followingthe feeding of beta-alanine and L-histidine (100 milligrams per kilogrambody weight and 12.5 milligrams per kilogram body weight, respectively,three times per day) over a period of 30 days.

FIGS. 3a, 3b, 3c, 3d, 3e and 3f are graphs depicting the contrast in thechanges in the concentrations of beta-alanine in blood plasma of sixhorses, before and at hourly intervals following the feeding ofbeta-alanine and L-histidine, as described in detail, below.

FIGS. 4a, 4b, 4c, 4d, 4e and 4f are graphs depicting the contrast in thechanges in the concentrations of L-histidine in blood plasma of sixhorses, before and at hourly intervals following the feeding ofbeta-alanine and L-histidine, as described in detail, below.

FIG. 5 is a graph depicting the contrast in the changes in the meanconcentrations of beta-alanine in equine blood plasma (n=6), before andat hourly intervals following the feeding of beta-alanine andL-histidine, as described in detail, below.

FIG. 6 is a graph depicting the contrast in the changes in the meanconcentrations of L-histidine in equine blood plasma (n=6), before andat hourly intervals following the feeding of beta-alanine andL-histidine (100 milligrams per kilogram body weight and 12.5 milligramsper kilogram body weight, respectively, three times per day) on thefirst and last day of a 30 day period of dietary supplementation.

FIG. 7 is a graph depicting the correlation between the increase in 6thoroughbred horses in the carnosine concentration in type II skeletalmuscle fibers (the average of the sum of type IIA and IIB fibers) andthe increase, between the 1st and 30th day of supplementation, in thearea under the blood plasma beta-alanine concentration-time curve overthe first 12 hours of the day (AUC_((0-12 hr))).

FIG. 8 is graph depicting the mean results of the administration ofbeta-alanine, broth, or carnosine to test subjects.

FIG. 9 is a graph depicting mean changes in plasma beta-alanine overnine hours of treatment.

FIG. 10 is a graph depicting the mean changes in plasma beta-alanineover 9 hours following the oral ingestion of 10 milligrams per kilogrambody weight of beta-alanine.

FIG. 11 is a graph depicting the mean (n=6) plasma beta-alanineconcentration over the 24 hours of Day 1 and Day 30 of the treatmentperiod.

FIG. 12 is a graph depicting changes in muscle carnosine concentrationpre and post treatment in different subjects. The red circles indicatethe muscle concentrations prior to supplementation.

FIG. 13 is a graph depicting muscle concentration (mean+SD) of carnosinebefore and post supplementation in three different treatment groups.

FIG. 14 is a graph depicting muscle concentration (mean+SD) of histidinebefore and post supplementation in three different treatment groups.

FIG. 15 is a graph illustrating data showing muscle concentration(mean+SD) of taurine before and post supplementation in four differenttreatment groups.

FIG. 16 is a graph illustrating data showing muscle concentration(mean+SD) of taurine before and post supplementation in differentsubjects.

FIG. 17 illustrates a table of data, described in detail as Table 9,below.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The invention provides compositions comprising beta-alanine, peptides ofbeta-alanine, analogues and derivatives thereof, beta-alanylhistidinedipeptides (e.g., carnosine, anserine, and balenine) and methods usingthese compositions for increasing the anaerobic working capacity of atissue comprising providing an amount of beta-alanine to blood or bloodplasma effective to increase beta-alanylhistidine dipeptide synthesis ina tissue. Beta-alanylhistidine dipeptides can include peptides ofbeta-alanine, such as carnosine, anserine, and balenine. In one aspect,they can have pKa values between approximately 6.8 and 7.1. In oneaspect, they can be involved in the regulation of intra-cellular pHhomeostasis during muscle contraction and the development of fatigue.The content of other substances involved in hydronium ion buffering,such as amino acid residues in proteins, inorganic and organicphosphates and bicarbonate, can be constrained by their involvement inother cell functions. In one aspect, the beta-alanylhistidine dipeptidesprovide an effective way of accumulating pH-sensitive histidine residuesinto a cell. Variations in the muscle beta-alanylhistidine dipeptideconcentrations affect the anaerobic work capacity of individualathletes.

The beta-alanylhistidine dipeptides are synthesized within the body frombeta-alanine and L-histidine. These precursors can be generated withinthe body or are made available via the diet, including from thebreakdown of an ingested beta-alanylhistidine dipeptide. Within thebody, beta-alanine is transported to tissues such as muscle. In atypical fed state, the concentration of beta-alanine is low incomparison with the concentration of L-histidine in human and equineblood plasma. These concentrations should be viewed in relation to theaffinity of the carnosine synthesizing enzyme, carnosine synthetase, forits substrates as determined by the Michaelis-Menten constant (Km). TheKm for histidine is about 16.8 μM. The Km for beta-alanine is betweenabout 1000 and 2300 μM. The low affinity of carnosine synthetase forbeta-alanine, and the low concentration of beta-alanine in muscle,demonstrate that the concentration of beta-alanine in muscle is limitingto the synthesis of the beta-alanylhistidine dipeptides.

Increasing the amount of beta-alanylhistidine dipeptides within a musclefavorably affects muscular performance and the amount of work that canbe performed by the muscle. Accordingly, it is desirable to increase thesynthesis and accumulation of beta-alanylhistidine dipeptides in atissue in a human or animal body.

The synthesis and accumulation of beta-alanylhistidine dipeptides in ahuman or animal body can be increased by increasing creatine within thebody, increasing the blood or blood plasma concentrations ofbeta-alanine, increasing the blood or blood plasma concentrations ofbeta-alanine and creatine, or increasing the blood or blood plasmaconcentrations of beta-alanine, L-histidine, and creatine. The increasein dipeptides can be simultaneous with the increase in beta-alanineconcentrations.

In one aspect, the compositions and methods of the invention can be usedto increase blood plasma concentrations of beta-alanine, L-histidineand/or creatine by ingestion or infusion of beta-alanine, peptides ofbeta-alanine, L-histidine, creatine, carnosine, anserine, and/orbalenine and/or active derivatives or analogs thereof alone or invarious combinations. The compositions of the invention can beadministered orally, enterally, or parenterally. For example,compositions of the invention can be orally ingested or infused throughthe skin through a topical cream or a patch.

The composition can include carbohydrates (e.g., simple carbohydrates),insulin, or agents that stimulate the production of insulin.Compositions can also include glycine, insulin, insulin mimics, and/orinsulin-action modifies.

The compositions can be a dietary supplement that can be ingested,injected, or absorbed through the skin. Preferably, the compositions canbe administered in one or more doses per day. The beta-alanine dosagecan be between about 1 milligram and about 200 milligrams per kilogrambody weight or the dose of a peptide of beta-alanine (e.g. carnosine)from 2.5 milligrams to 500 milligrams per kilogram body weight. In oneaspect, the total amount of beta-alanine (or other composition of theinvention) administered can be at least 200 mg, from 200 mg to 5 g, orfrom 5 g or more per day for a human. A single dose of activeingredient, e.g., beta-alanine, carnosine, anserine, or balenine, ormixtures thereof, may be formulated to be in the amount about 200, 400,800 mg or more. The creatine (e.g., creatine monohydrate) dosage, ordosage of other compositions of the invention, can be between about 5milligrams to 200 milligrams per kilogram body weight. The L-histidinedosage, or dosage of other compositions of the invention, can be betweenabout 1 milligram to 100 milligrams per kilogram body weight. The simplecarbohydrate (e.g., glucose) dosage, or dosage of compositions of theinvention, can be between about 0.5 and 2.0 grams per kilogram bodyweight.

In an 80 kilogram person, suitable dosages per day can be between 0.08grams to 16.0 grams of beta-alanine or 200 milligrams to 40 grams of apeptide of beta-alanine, 0.4 grams to 16.0 grams of creatinemonohydrate, 0.08 grams to 8.0 grams of L-histidine, or 40 grams to 160grams of glucose or other simple carbohydrate. The composition can be ina solid form or a liquid form or in a suspension which can be ingestedor infused into the body. The composition can be ingested by humans inan amount of between 0.08 grams and 1000 grams or more per day, whichmay be taken in one or more parts throughout the day. In animals, thedaily intake will be adjusted by body weight.

In one aspect, the total amount of a peptide of beta-alanine, forexample, carnosine, anserine or balenine that can be administered perday may be at least 500 mg, between about 500 mg to about 5 g, betweenabout 5 g to about 16 g, or greater than 16 g. A single dose of apeptide of beta-alanine creatine, anserine or balenine, or mixturesthereof, may be formulated to be in the amount of 0.5, 1, 1.5, or 2 g(for each, or all, or the peptides in a formulation comprising amixture).

For humans and animals, the compositions can be:

-   -   (a) 1% to 99% by weight of beta-alanine or 1% to 99% by weight        of a peptide of beta-alanine;        -   1% to 99% by weight of creatine monohydrate; and        -   0% to 98% by weight of water;    -   (b) 1% to 98% by weight of beta-alanine or 1% to 98% by weight        of a peptide of beta-alanine;        -   1% to 98% by weight of L-histidine;        -   1% to 98% by weight of creatine monohydrate; and        -   0% to 97% by weight of water;    -   (c) 1% to 20% by weight of beta-alanine or 1% to 20% by weight        of a peptide of beta-alanine;        -   39% to 99% by weight of glucose or other simple            carbohydrate; and        -   0% to 60% by weight of water; or    -   (d) 1% to 20% by weight of beta-alanine or 1% to 20% by weight        of a peptide of beta-alanine;        -   1% to 20% by weight of L-histidine;        -   39% to 99% by weight of glucose or other simple            carbohydrate; and

0% to 60% by weight of water.

In one aspect, compositions are applied to a body for at least threedays, from 3 days to 2 weeks, from 2 weeks to 4 weeks, or longer. Incertain regimens, the daily dosages are gradually increased ordecreased. This can be done daily, every couple of days, or weekly.

EXAMPLES

The following are specific examples of the methods and compositions forincreasing the anaerobic working capacity of muscle and other tissues.

Example 1

The effect of supplementation of a normal diet with multiple daily dosesof beta-alanine and L-histidine on the carnosine concentration in typeI, IIA, and JIB skeletal muscle fibers of thoroughbred horses wasassessed. Six experimental thoroughbred horses of normal health (threefillies and three geldings), aged 4 to 9 years, underwent one month (30days) of dietary conditioning (pre-supplementation period) prior to thecommencement of the supplementation period. During the dietaryconditioning period each horse was fed a diet comprising 1 kilogram ofpelleted feed (Spillers racehorse cubes) and 1 kilogram of soaked sugarbeet pulp as a source of complex and simple carbohydrates, three timesper day (at 08:30, 12:30, and 16:30, respectively). Soaked hay (3kilograms dry weight) was also provided twice daily (at 09:00 and17:00). Water was provided ad libitum.

During the supplementation period, an identical feeding regime wasimplemented. However, each hard feed meal was supplemented withbeta-alanine and L-histidine (free base). Beta-alanine and L-histidinewere mixed directly into the normal feed. Individual doses ofbeta-alanine and L-histidine were calculated according to body weight.Beta-alanine was administered at 100 milligrams per kilogram body weightand L-histidine at 12.5 milligrams per kilogram body weight. Dietarysupplementation was begun on day 1 of the protocol and discontinued atthe end of day 30. Heparinized blood samples (5 milliliters) werecollected on days 1, 6, 18, 24, and 30. On day 1 and day 30, bloodsamples were collected prior to the first feed and at hourly intervalsfor a total of 12 hours each day. On the three intervening samplingdays, blood was collected prior to the first feed and 2 hours after eachsubsequent feed. On the day before the start of supplementation (day 0)a muscle biopsy was taken, following application of local anesthesia ofthe skin, from the right middle gluteal muscle (m. gluteus medius) ofeach horse using a Bergstrom-Stille percutaneous biopsy needle.Subsequent muscle biopsies were collected immediately after the end ofthe supplementation period (day 31) as close as possible to the originalsampling site. Clinical monitoring of the horses was performed daily.This comprised a visual examination and measurement of body weight,twice-daily measurement of rectal temperature, and weekly blood samplingfor clinical biochemistry and hematology. During the course of thestudy, the horses received no formal training or exercise, although theywere allowed one hour of free exercise each day.

Fragments of individual muscle fibers dissected from freeze-dried musclebiopsies were characterized as either type I, IIA or IIB byhistochemical staining for myosin ATPase activity at pH 9.6 followingpre-incubation at pH 4.5 by a modification of the method described in,Kaiser and Brook, Arch. Neural., 23:369-379 (1970).

Heparinized blood plasma samples were extracted and analyzed forbeta-alanine and L-histidine concentrations by high-performance liquidchromatography (HPLC). Individual weighed muscle fibers were extractedand analyzed for carnosine by HPLC according to the method described in,Dunnett and Harris, “High-performance liquid chromatographicdetermination of imidazole dipeptides, histidine, 1-methylhistidine and3-methylhistidine in muscle and individual muscle fibers,” J.Chromatogr. B. Biomed. Appl., 688:47-55 (1997).

Differences in carnosine concentrations within fiber types before andafter supplementation were established within horses using one-wayanalysis of variance (ANOVA). In instances where differences weredetected, significance was determined using a multiple comparison test(Fisher's PLSD).

No palatability problems were encountered with the addition ofbeta-alanine and L-histidine to the feed. No adverse physiological orbehavioral effects of the supplemented diet were observed in any of thehorses during the thirty days of supplementation. No significant changesin body weight were recorded, and rectal temperatures remained withinthe normal range. No acute or chronic changes in clinical biochemistryor hematology were observed. Beta-alanine was not detected in the plasmaof any of the horses prior to the start of supplementation. The lowerlimit of quantitation for beta-alanine in plasma by the assay used was 3micromolar (μM). Plasma L-histidine concentrations in the six horsesprior to the start of supplementation were between 36.6 and 54.4 μM.

Individual changes in blood plasma beta-alanine and L-histidineconcentrations for five of the six horses over all the sampling days areshown in FIGS. 1 and 2, respectively. There was a trend towards anincrease in the pre-feeding concentrations of blood plasma beta-alanineand L-histidine with increasing time of supplementation. Furthermore,over the thirty day supplementation period, the blood plasmaconcentration response to supplementation was also increased. Theresponse was greater for beta-alanine.

Comparisons of the changes in blood plasma beta-alanine and L-histidineconcentrations prior to the first feed of the day, and hourly thereafterbetween the first and last days of the supplementation period, for thesix individual horses, are shown in FIGS. 3a and 3b , and FIGS. 4a and4b , respectively. FIGS. 3a, 3b, 3c, 3d, 3e and 3f are graphs depictingthe contrast in the changes in the concentrations of beta-alanine inblood plasma of six horses, before and at hourly intervals following thefeeding of beta-alanine and L-histidine (100 milligrams per kilogrambody weight and 12.5 milligrams per kilogram body weight, respectively,three times per day) on the first and last day of a 30 day period ofdietary supplementation. FIGS. 4a, 4b, 4c, 4d, 4e and 4f are graphsdepicting the contrast in the changes in the concentrations ofL-histidine in blood plasma of six horses, before and at hourlyintervals following the feeding of beta-alanine and L-histidine (100milligrams per kilogram body weight and 12.5 milligrams per kilogrambody weight, respectively, three times per day) on the first and lastday of a 30 day period of dietary supplementation. FIG. 5 is a graphdepicting the contrast in the changes in the mean concentrations ofbeta-alanine in equine blood plasma (n=6), before and at hourlyintervals following the feeding of beta-alanine and L-histidine (100milligrams per kilogram body weight and 12.5 milligrams per kilogrambody weight, respectively, three times per day) on the first and lastday of a 30 day period of dietary supplementation. The mean (SD) changes(n=6) in blood plasma beta-alanine concentration over time during the 24hours of the first (day 1) and last (day 30) days of the supplementationperiod are contrasted in FIG. 5. The area under the mean blood plasmabeta-alanine concentration versus time curve over 24 hours(AUC_((0-24hr))) was much greater on day 30 of the supplementation.

The mean (SD) changes (n=6) in blood plasma L-histidine concentrationover time during the 24 hours of the first (day 1) and last (day 30)days of the supplementation period are contrasted in FIG. 6. The areaunder the mean blood plasma beta-alanine concentration vs. time curveover 24 hours (AUC_((0-24hr))) was greater on day 30 of thesupplementation. The greater AUC for blood plasma beta-alanine on thelast day of supplementation (day 30) in contrast to the first day ofsupplementation (day 1) suggests the increased uptake of beta-alaninefrom the equine gastro-intestinal tract with progressivesupplementation. A similar effect was observed for changes in bloodplasma L-histidine concentration during the supplementation period. Peakblood plasma concentrations of beta-alanine and L-histidine occurredapproximately one to two hours post-feeding in each case.

A total of 397 individual skeletal muscle fibers (192pre-supplementation; 205 post-supplementation) from the six horses weredissected and analyzed for carnosine. Mean (SD) carnosine concentration,expressed as millimoles per kilogram dry weight (mmol kg⁻¹ dw), in pre-and post-supplementation type I, IIA, and IIB skeletal muscle fibersfrom the six individual horses are given in Table 1 where n is thenumber of individual muscle fibers analyzed. Following thirty days ofbeta-alanine and L-histidine supplementation the mean carnosineconcentration was increased in type IIA and IIB fibers in all sixhorses. These increases were statistically significant in seveninstances. The increase in mean carnosine concentration in type IIBskeletal muscle fibers was statistically significant in five out of sixhorses. The increase in mean carnosine concentration in type IIAskeletal muscle fibers was statistically significant in two out of sixhorses.

TABLE 1 Horse Day Type 1 n Type IIA n Type IIB n 6 0 32.3 (14.5) 3 72.1(47.7) 11 111.8 (22.8) 14 31 16.2 (20.9) 17 117.7 (38.7) 12 5 0 59.5(3.9) 2 102.6 (12.7) 12 131.2 (26.6) 26 31 55.5 1 112.2 (17.1) 18 153.3(28.0)** 22 4 0 44.8 (6.6) 4 59.9 (19.5) 13 108.6 (41.5) 19 31 37.0(9.3) 2 88.0 (34.2)* 17 152.4 (65.0)* 19 1 0 56.7 (5.3) 2 88.5 (20.9) 15101.3 (15.2) 13 31 57.8 1 96.1 (17.3) 19 14.3 (13.3)* 11 2 0 — 4 89.6(16.2) 13 104.2 (22.2) 14 31 65.9 (13.2) 102.2 (22.1) 18 142.0 (35.4)***12 3 0 30.9 (4.0) 2 85.1 (20.3) 6 113.5 (20.4) 23 31 — 105.0 (17.6)* 23135.4 (24.9)* 9 Mean 0 44.8 13 83.0 70 111.8 109 31 54.1 8 96.6* 112135.9** 85 *significantly different to pre-supplementation, p < 0.05**significantly different to pre-supplementation, p < 0.01***significantly different to pre-supplementation, p < 0.005

The absolute (e.g. mmol kg⁻¹ dw) and percentage increases in the meancarnosine concentrations in type HA and IIB skeletal muscle fibers fromthe six horses are listed in Table 2.

TABLE 2 Type IIA Type IIB Absolute Type IIA Absolute Type IIB Horseincrease % increase increase % increase 6 4.1 5.7 5.6 5.3 5 9.6 9.4 22.116.8 4 28.1 46.9 43.8 40.3 1 7.6 8.6 13.0 12.8 2 12.6 14.1 37.8 36.3 319.9 23.4 21.9 19.3 Mean 13.6 18.0 24.1 21.8

It was observed that the individual horses which showed the greaterincrease in muscle carnosine concentration following thirty days ofsupplementation also demonstrated the greater increase in blood plasmabeta-alanine AUC between day 1 and day 30 of the supplementation period.Referring to FIG. 7, a significant correlation (r=0.986, p<0.005) forfive of the six horses was observed between the increase in meancarnosine concentration, averaged between type IIA and IIB skeletalmuscle fibers and the increase, between the 1st and 30th day ofsupplementation, in blood plasma beta-alanine AUC, over the first 12hours (AUC_((0-12hr))). Only five horses were used to calculate theregression line. Horse 6 (filled circle) showed no appreciable increasein blood plasma beta-alanine concentration greater than that observed onday 1 until the last day of supplementation. This was unlike the otherfive horses, which showed a progressive increase with each sampling day.For this reason horse 6 was excluded from the calculation of theregression equation.

Increases in muscle carnosine concentration following thirty days ofsupplementation with beta-alanine and L-histidine will cause a directincrease in total muscle buffering capacity. This increase can becalculated by using the Henderson-Hasselbach Equation. Calculated valuesfor the increases in muscle buffering capacity in type IIA and IIBskeletal muscle fibers in the six thoroughbred horses are shown in Table3.

TABLE 3 Type Type Type Type IIA Type Type IIB IIA IIA Δβmtotal IIB IIBΔβmtotal Horse Day βmcar βmtotal (%) βmcar βmtotal (%) 6 0 23.9 93.9+1.5 37.1 107.1 +1.8 31 25.3 95.3 39.0 109.0 5 0 34.0 104.0 +3.1 43.5113.5 +6.4 31 37.2 107.2 50.8 120.8 4 0 19.9 89.9 +10.3 36.0 106.0 +13.731 29.2 99.2 50.5 120.5 1 0 29.3 99.3 +2.6 33.6 103.6 +4.2 31 31.9 101.937.9 107.9 2 0 29.7 99.7 +4.2 34.5 104.5 +12.1 31 33.9 103.9 47.1 117.13 0 28.2 98.2 +6.7 37.6 107.6 +6.8 31 34.8 104.8 44.9 114.9 Mean 0 27.597.5 +4.7 37.1 107.1 +7.5 31 32.1 102.1 45.0 115.0

Example 2

The effect of supplementation of a normal diet with single and multipledaily doses of beta-alanine in free or peptide bound form on thebeta-alanine and beta-alanyl dipeptide concentrations of plasma ofhumans was assessed. The plasma concentration of beta-alanine in sixnormal subjects following the consumption of a broth deliveringapproximately 40 milligrams per kilogram body weight of beta-alanine wasmonitored. Doses of 10 and 20 milligrams per kilogram body weight ofbeta-alanine were also given.

The broth was prepared as follows. Fresh chicken breast (skinned andboned) was finely chopped and boiled for fifteen minutes with water (1liter for every 1.5 kg of chicken). Residual chicken meat was removed bycourse filtration. The filtrate was flavored by the addition of carrot,onion, celery, salt, pepper, basil, parsley and tomato puree, andreboiled for a further fifteen minutes and then cooled before finalfiltration through fine muslin at 4° C. The yield from 1.5 kilograms ofchicken and one liter of water was 870 mL of broth. A portion of thestock was assayed for the total beta-alanyl-dipeptide content (e.g.,carnosine and anserine) and beta-alanine. Typical analyses were:

total beta-alanyl-dipeptides 74.5 mM  free beta-alanine 5.7 mM

The six male test subjects were of normal health and between 25-53 yearsof age, as shown in Table 4. The study commenced after an overnight fast(e.g., a minimum of 12 hours after the ingestion of the last meatcontaining meal). Subjects were given the option to consume a smallquantity of warm water prior to the start of the study. Catheterizationwas begun at 08:30 and the study started at 09:00.

As a control, 8 milliliters per kilogram body weight of water wasingested (e.g., 600 mL in a subject weighing 75 kilograms).

In one session, 8 milliliters per kilogram body weight of brothcontaining approximately 40 milligrams per kilogram body weight ofbeta-alanine (e.g., in the form of anserine and carnosine) was ingested.For a subject weighing 75 kilograms, this amounted to the ingestion of600 milliliters of broth containing 3 grams of beta-alanine. In anothersession, 3 milliliters per kilogram body weight of a liquid containingthe test amount of beta-alanine with an additional 5 milliliters perkilogram body weight of water was ingested. In all sessions, subjectsadditionally consumed a further 8 milliliters per kilogram body weightof water (in 50 mL portions) during the period of 1 to 2 h afteringestion. A vegetarian pizza was provided after 6 hours. An ordinarydiet was followed after 8 hours.

2.5 milliliter venous blood samples were drawn through an indwellingcatheter at 10 minute intervals for the first 90 minutes and then after120, 180, 240 and 360 minutes. The blood samples were dispensed intotubes containing lithium-heparin as an anti-coagulant. The catheter wasmaintained by flushing with saline. Plasma samples were analyzed by HPLCaccording to the method described in Jones & Gilligan (1983) J.Chromatogr. 266:471-482 (1983).

Table 4 summarizes the allocation of treatments during the beta-alanineabsorption study. The estimated equivalent doses of beta-alanine arepresented in Table 4.

TABLE 4 Broth β-ala β-ala β-ala β-ala Carnosine Age Weight 40 mg/kg 0mg/kg 10 mg/kg 20 mg/kg 40 mg/kg 20 mg/kg Subject yrs kg bwt bwt bwt bwtbwt bwt 1 53 76 + + + + 2 33 60 + + + 3 29 105 + + + + 4 31 81 + + + + 530 94 + + + + 6 25 65 + + + +

Plasma concentration curves following each treatment are depictedgraphically in FIG. 8. Mean results of the administration ofbeta-alanine, broth, or carnosine according to the treatments schedulein Table 4. Plasma beta-alanine was below the limit of detection in allsubjects on the control treatment. Neither carnosine or anserine weredetected in plasma following ingestion of the chicken broth or any ofthe other treatments. Ingestion of the broth resulted in a peakconcentration in plasma of 427.9 (SD 161.8) μM. Administration ofcarnosine equivalent to 20 milligrams per kilogram body weight ofbeta-alanine in one test subject resulted in an equivalent increase inthe plasma beta-alanine concentration.

Administration of all treatments except control resulted in an increasein the plasma taurine concentration. The changes in taurineconcentration mirrored closely those of beta-alanine. Administration ofbroth, a natural food, caused an equivalent increase in plasma taurine,indicating that the response occurs normally following the ingestion ofmost meals.

Example 3

The effect of administration of three doses of 10 milligrams perkilogram body weight of beta-alanine per day (i.e., administered in themorning, noon, and at night) for seven days on the plasma concentrationprofiles of beta-alanine and taurine were investigated. The plasmaconcentration profiles following administration of 10 milligrams perkilogram body weight of beta-alanine were studied in three subjects atthe start and end of a seven-day period during which they were giventhree doses of the beta-alanine per day.

Three male subjects of normal health, aged between 33-53 years werestudied. Test subjects received three doses per day of 10 milligrams perkilogram body weight of beta-alanine for eight days. In two subjects,this was followed by a further 7 days (days 9-15) when three doses of 20milligrams per kilogram body weight per day were given. Subjectsreported at 8 am to the blood collection laboratory on days 1 (prior toany treatment given), 8 and 15 following an overnight fast. Subjectswere asked not to consume any meat containing meal during the 12 hourspreceding the study. On each of these three test days subjects werecatheterized and an initial blood sample taken when the beta-alanine wasadministered at or close to 9 am, 12 noon, and 3 pm. Blood samples weredrawn after 30, 60, 120 and 180 minutes, and analyzed for changes in theplasma concentration of beta-alanine and taurine. 24-hour urine sampleswere collected over each day of the study and analyzed by HPLC todetermine the excretion of beta-alanine and taurine. The treatments aresummarized in Table 5.

TABLE 5 Treatment Day Day 1 Day 8 Day 15 beta-alanine 10 mg/kg bwt 10mg/kg bwt 20 mg/kg bwt 1 + + + 2 + + + 3 + +

The plasma beta-alanine concentrations are summarized in FIG. 9. Eachdose resulted in a peak beta-alanine concentration at one-half hour orone hour after ingestion followed by a decline to a 0-10 micromolarbasal level at three hours, just prior to administration of the nextdose. The response on day 8 of the treatment tended to be less than onday 1, as indicated by the area under the plasma concentration curve.

Example 4

The effect of administration of three doses of 40 milligrams perkilogram body weight of beta-alanine per day (i.e., administered in themorning, noon, and at night) for 2 weeks on the carnosine content ofmuscle and isometric endurance at 66% of maximal voluntary contractionforce was investigated.

Six normal male subjects, aged 25 to 32 years, that did not haveevidence of metabolic or muscle disease were recruited into the study.The subjects were questioned regarding their recent dietary andsupplementary habits. None of subjects was currently taking supplementscontaining creatine, or had done so in recent testing supplementationprocedures. The physical characteristics of the test subjects aresummarized in Table 6.

TABLE 6 Subject Age (years) Weight (kg) 1 29 78 2 31 94 3 29 105 4 25 655 31 81 6 25 75 7 53 76

Two days before treatment, a preliminary determination of maximalvoluntary (isometric) contraction force (MVC) of knee extensors with thesubject in the sitting position was carried out. MVC was determinedusing a Macflex system with subjects motivated by an instantaneousvisual display of the force output. For each subject, two trials werecarried out to determine endurance at 66% MVC sustained until the targetforce could no longer be maintained despite vocal encouragement. Thisfirst contraction was subsequently followed by a rest period of 60seconds, with the subject remaining in the isometric chair. After therest period, a second contraction was sustained to fatigue. Following asecond rest of 60 seconds, a third contraction to fatigue wasundertaken.

One day before treatment, the subjects reported to the isometric testlaboratory between 8 and 10 am. MVC was determined and endurance at 66%MVC over three contractions with 60 second rest intervals, as describedabove, was determined. Measurements were determined using the subject'sdominant leg. A biopsy of the lateral portion of the vastus lateraliswas taken again from the dominant leg.

On day 1 of the treatment study, subjects reported to the blood samplinglaboratory at 8 am following an overnight fast and a minimum of 12 hourssince the last meat containing meal. Following catheterization and abasal blood sample, each subject followed the supplementation and bloodsampling protocol described in Example 3. A dose of 10 milligrams perkilogram body weight of beta-alanine was administered at time 0 (9 am),3 hours, and 6 hours.

On days 2-15, subjects continued to take three doses of 10 milligramsper kilogram body weight of beta-alanine.

In the morning of day 14, post-treatment isometric exercise tests wereconducted on the dominant leg to determine MVC and endurance at 66% MVCrelative to the 66% MVC measured on the day prior to treatment. In theafternoon, a muscle biopsy was taken of the vastus lateralis from closeto the site of the biopsy taken on the day before treatment.

On day 15, the procedures followed on day 1 were repeated to determineany overall shift in the plasma concentration profile of beta-alanineand taurine over the 15 days of supplementation. Mean changes in plasmabeta-alanine over 9 hours following the oral ingestion of 10 milligramsper kilogram body weight of beta-alanine at 0, 3 and 6 hours on days 1and 15 when dosing at 3×10 milligrams per kilogram body weight per dayare shown in FIG. 10.

One additional test subject (number 7) followed the study, taking threedoses 10 milligrams per kilogram body weight for 7 days followed bythree doses of 20 milligrams per kilogram body weight for 7 days. Nomuscle biopsies were taken from this test subject.

There was no apparent change in the muscle carnosine content in themuscle of the six subjects biopsied. Changes in plasma taurineconcentrations in the six subjects mirrored those of beta-alanine, asnoted in Example 2.

Values from the MVC and endurance at 66% MVC measurements one day beforetreatment and after 14 days after treatment with three doses of 10milligrams per kilogram body weight of beta-alanine are listed in Table7. The mean endurance time at 66% MVC increased in 5 of the 6 subjects.An increase was also seen in subject 7 who had taken the higher dose.

TABLE 7 time @ 66% time @ 66% time @ 66% Total MVC 1st MVC 2nd MVC 1stMVC 2nd MVC 3rd Contraction Subject try N try N seconds seconds secondsTime seconds Pre 1 784.5 821.9 48.53 29.03 23.78 100.83 2 814.4 886.248.40 26.03 16.90 91.33 3 984.9 970.4 38.15 26.03 16.78 80.95 4 714.6740.4 89.03 56.15 45.65 190.83 5 1204.8 1217.2 37.65 27.64 21.53 86.83 6722.4 716.8 46.78 29.40 21.90 98.08 Pre mean 870.9 892.1 51.4 32.4 24.3108.1 Pre SD 190.6 184.6 19.1 11.7 10.8 41.2 Post 1 895.6 908.0 47.0830.38 24.03 101.48 2 832.2 908.0 46.65 31.28 18.40 96.33 3 973.7 952.242.65 25.03 16.03 83.70 4 814.1 863.9 114.40 64.28 48.53 227.20 5 1246.61233.0 42.03 22.78 19.40 84.20 6 760.8 773.3 52.28 31.53 25.95 109.73Post mean 920.5 939.7 57.5 34.2 25.4 117.1 Post SD 175.7 156.0 28.1 15.211.9 54.9 Subject 7 Pre 858.18 861.54 54.0 Post 792.54 851.41 62.0

Example 5

The effect of 4 weeks of beta-alanine supplementation using two dosingregimens and an isomolar dose of L-carnosine (beta-alanylhistidine)administered over 4 weeks on the muscle carnosine content wereinvestigated.

Fifteen male subjects, aged 20 to 29 years with no obvious signs ofclinical disease and with heights and weights within the normal range,were recruited into the study (Table 8). All subjects participated inone or more sports and all ate a mixed diet containing variable amountsof meat. A record of each subject's approximate intake of meat duringthe course of the investigation was made.

TABLE 8 Summary of subjects' physical characteristics for each of thethree treatment groups: AGE HEIGHT MASS Treatment Mean ± SD Mean ± SDMean ± SD 1 24.4 ± 2.7 182.3 ± 7.5 80.0 ± 15.9 2 23.8 ± 1.9 180.9 ± 5.480.6 ± 8.6  3 24.0 ± 3.8 180.1 ± 3.8 80.4 ± 12.1

Five subjects were allocated to one of three treatment groups (1, 2, and3). During the study, their diet was supplemented with eitherbeta-alanine or carnosine as described in Table 9 (FIG. 17). Thesupplements were provided in soft gelatine capsules containing either400 mg beta alanine or 500 mg carnosine.

In Group 1, beta-alanine was administered in 4 separate doses throughoutthe day (qid) at a steady rate for four weeks.

In Group 2, beta-alanine was administered as 8 separate doses throughoutthe day, rather than as 4 doses, in an attempt to maintain a more evenincrease in the blood-plasma concentration. In addition, the dose wasincreased progressively each week by 800 mg per day.

In Group 3, carnosine was administered at approximately the sameisomolar dose as in Group 2, again divided into 8 doses. This treatment,therefore, contained approximately the same amount of beta-alanine as inGroup 2, when hydrolyzed to its constituent amino acids.

The subjects took the supplements at the times indicated in Table 9(FIG. 17). A single muscle biopsy of the vastus lateralis was takenbefore and at the end of the supplementation using the percutaneousneedle biopsy procedure of Bergström (1962). In brief, the procedureinvolves the insertion of a hollow bored needle under local anestheticand sterile conditions to obtain specimens around 20-40 mg containingapproximately 100-700 muscle fibers. The skin and subcutaneous tissue isanesthetized with 1% lignocaine (avoiding contact with the muscle). Anincision is made to the skin and deep fascia with a scalpel blade. Theneedle minus central rod is inserted into the muscle. The muscle bulk ispressed into a needle side-window. A sample is cut by ramming an inner,sharpened cylinder along the needle. The needle is removed and thecentral rod is used to evacuate the specimen. The wound is then closed.

Muscle samples from the subjects were frozen in liquid nitrogen,freeze-dried and analyzed for muscle carnosine and taurine contents byHPLC. Table 9 (FIG. 17) shows a breakdown of the dosing strategiesemployed in each of the three treatment groups. Results: There was nochange in body mass in either beta-alanine or carnosine supplementedsubjects.

Changes in Muscle Carnosine (Table 10 and FIGS. 12 and 13).

A significant increase in muscle carnosine content was recorded for thesubjects in Groups 1 and 3. In Group 2, one subject (no. 10) with thehighest initial carnosine content (initial carnosine content: 33.3mmol/kg dry muscle) showed no change in his muscle carnosine content(post content: 33.7 mmol/kg dm). When this subject was deleted fromGroup 2, this group showed a significant increase of the same order asseen in the other Groups. Subject 10 was a medium to high consumer ofmeat and otherwise unremarkable.

Supplementation with either beta-alanine or carnosine at the same dose(Groups 2 and 3) appeared to be equally effective in increasing themuscle carnosine content.

The pattern of change is reminiscent of the changes observed withcreatine loading and may suggest that there is a threshold which isquickly reached, with further supplementation having no further effect.In the case of subject 10, while not wishing to be bound by this theory,a threshold appears to have been reached even before the start ofsupplementation. However, there are exceptions to the notion of an upperthreshold, notably subjects 6 (post supplementation carnosine: 45.9mmol/kg dry muscle) and 15 (post supplementation carnosine: 68.9 mmol/kgdm). Subjects 6 and 15 were unremarkable in either their dietarypatterns or participation in physical exercise.

Table 10 is a summary of data for carnosine muscle concentrations fortreatment Groups 1 to 3. Treatment Group 2, in italics, is withoutsubject 10 who did not exhibit an increase in muscle carnosineconcentration. The initial carnosine concentration in subject 10 was thehighest of all subjects and may have already been at an “upperthreshold” level prior to supplementation.

TABLE 10 Treatment 1 2 2 3 n = 5 n = 5 n = 4 n = 5 Mean pre 19.58 24.2321.96 23.15 SD pre 3.71 5.27 1.64 5.07 Mean post 27.38 35.27 35.67 39.52SD post 2.96 6.18 7.08 16.95 Mean difference 7.80 11.04 13.72 16.37 SDdifference 0.81 9.20 8.08 12.06 Sign *** ns * * Min difference 6.99 0.358.62 7.05 Max difference 9.08 25.77 25.77 37.39 Mean % change 42.1 51.664.2 65.8 SD % change 14.9 46.5 42.7 31.8 Min % change 31.5 1.1 41.038.2 Max % change 68.0 128.2 128.2 118.7

Changes in Muscle Beta-Alanine and Histidine (Table 11 and FIG. 14)

The muscle beta-alanine concentration was below the limit of detection(<0.2 mmol/kg dm) before and at the end of supplementation. In somesubjects, a final dose of beta-alanine was taken within 1 to 2 hours ofthe final muscle biopsy.

There was no change in the muscle histidine concentration withsupplementation with either beta-alanine or carnosine, the latter havingthe potential to release histidine into the general circulation. Therewas no decrease in the histidine concentration in response to theincreased synthesis of carnosine (each mole requiring one mole ofhistidine).

Changes in Muscle Taurine (Table 12 and FIG. 15 and FIG. 16)

While beta-alanine at high concentrations may interfere with the uptakeof taurine into tissues, previous observations show an increase in theplasma taurine concentration and loss of taurine in urine following bothbeta-alanine and carnosine administration, no loss of muscle taurine wasnoted in this study in any of the three Groups. Marked changes in themuscle taurine content occurred in some individuals, but both increasesand decreases were observed. FIG. 15 is a graph illustrating datashowing muscle concentration (mean+SD) of taurine before and postsupplementation in four different treatment groups. FIG. 16 is a graphillustrating data showing muscle concentration (mean+SD) of taurinebefore and post supplementation in different subjects.

CONCLUSIONS

These studies demonstrate that the supplements of beta-alanine andcarnosine of the invention have the potential to increase the musclecarnosine content. Based on the test results, they appear to be equallyeffective in increasing carnosine in tissue.

The changes in the muscle buffering capacity help maintain theintracellular microenvironment during intense exercise, countering theaccumulation of H⁺. As such, supplementation with beta-alanine orcompounds delivering beta-alanine on ingestion may have a positiveeffect on exercise capacity in sports and those general daily activitiesleading to lactate accumulation. In view of the other chemicalactivities ascribed to carnosine (as an anti-oxidant and anti-glycatingagent), an increase in carnosine concentration may have other beneficialeffects apart from those arising from an increase in muscle bufferingcapacity.

Four weeks of supplementation did not result in any apparent loss oftaurine in the muscles.

TABLE 11 summary data for histidine muscle concentrations in treatmentGroups 1 to 3: Treatment 1 2 3 n = 5 n = 5 n = 5 mean pre 5.76 5.56 7.01SD pre 0.59 0.63 2.60 mean post 5.12 5.51 5.38 SD post 1.17 0.87 1.39mean diff −0.64 −0.05 −1.63 SD diff 1.24 0.70 2.87 Sign ns ns ns %change −10.59 −0.78 −17.47 SD % change 21.65 12.65 29.56

TABLE 12 Summary data for taurine muscle concentrations in treatmentsGroups 1 to 3: Treatment 1 2 3 n = 5 n = 5 n = 5 Mean pre 36.52 28.6835.40 SD pre 7.77 5.29 8.92 Mean post 33.70 27.54 32.32 SD post 7.988.77 15.19 Mean difference −2.82 −1.15 −3.08 SD difference 7.96 6.0413.61 Sign ns ns ns % change −6.28 −4.46 −7.66 SD % change 21.54 24.0634.97

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1.-9. (canceled)
 10. An improved method of delaying the onset of musclefatigue in a human supplementing in a 24 hour period over a period oftime with a human dietary supplement comprising beta-alanine that is notpart of a dipeptide, oligopeptide or polypeptide in an amount sufficientto increase beta-alanylhistidine synthesis in the muscle, wherein theimproved method comprises increasing the amount of beta-alanine in thedietary supplement ingested in a 24 hour period for a period of time,wherein an increased amount is provided at a first increased amount fora first period of time and then is provided at a second increased amountfor a second period of time, and wherein the ingested amount in the 24hour period is ingested in one serving in the 24 hour period or inmultiple servings at different times during the 24 hour period, andwherein the period of time includes more than two 24 hour periods. 11.The method of claim 10, wherein the amount of beta-alanine ingested isincreased daily.
 12. The method of claim 10, wherein the amount ofbeta-alanine ingested is increased every other day.
 13. The method ofclaim 10, wherein the amount of beta-alanine ingested is increasedweekly.
 14. The method of claim 10, wherein the amount of beta-alanineingested is increased every other week.
 15. The method of claim 10,wherein the amount of beta-alanine ingested is increased every day thatthe human participates in physical activity.
 16. The method of claim 10,wherein the amount of beta-alanine ingested is increased in 0.1 gincrements.
 17. The method of claim 10, wherein the amount ofbeta-alanine ingested is increased in 0.2 g increments.
 18. The methodof claim 10, wherein the amount of beta-alanine ingested is increased in0.3 g increments.
 19. The method of claim 10, wherein the amount ofbeta-alanine ingested is increased in 0.4 g increments.
 20. The methodof claim 10, wherein the amount of beta-alanine ingested is increased in0.5 g increments.
 21. The method of claim 10, wherein the amount ofbeta-alanine ingested is increased in 0.6 g increments.
 22. The methodof claim 10, wherein the amount of beta-alanine ingested is increased in0.7 g increments.
 23. The method of claim 10, wherein the amount ofbeta-alanine ingested is increased in 0.8 g increments.
 24. The methodof claim 10, wherein the amount of beta-alanine ingested is increased in0.9 g increments.
 25. The method of claim 10, wherein the amount ofbeta-alanine ingested is increased in 1.0 g increments.
 26. The methodof claim 10, wherein the amount of beta-alanine ingested is increased toa maximum of 16 g.
 27. The method of claim 10, wherein the period oftime is from 3 days to 8 weeks.
 28. The method of claim 27, where theperiod of time is from 5 days to 6 weeks.
 29. The method of claim 28,wherein the period of time is from 1 week to 4 weeks.
 30. The method ofclaim 10, wherein the improvement further comprises ingesting an amountof L-histidine to prevent loss of muscle histidine.
 31. The method ofclaim 30, wherein the amount of L-histidine is 0.1 g to 16 g.
 32. Themethod of claim 10, wherein the improvement further comprises decreasingthe amount of beta-alanine ingested in a 24 hour period for a period oftime following increasing the amount of beta-alanine ingested in a 24hour period for a period of time.
 33. The method of claim 32, whereinthe amount of beta-alanine ingested is decreased daily.
 34. The methodof claim 32, wherein the amount of beta-alanine ingested is decreasedevery other day.
 35. The method of claim 32, wherein the amount ofbeta-alanine ingested is decreased weekly.
 36. The method of claim 32,wherein the amount of beta-alanine ingested is decreased every otherweek.
 37. The method of claim 32, wherein the amount of beta-alanineingested is decreased every day that the human participates in physicalactivity.
 38. The method of claim 32, wherein the amount of beta-alanineingested is decreased in 0.1 g increments.
 39. The method of claim 32,wherein the amount of beta-alanine ingested is decreased in 0.2 gincrements.
 40. The method of claim 32, wherein the amount ofbeta-alanine ingested is decreased in 0.3 g increments.
 41. The methodof claim 32, wherein the amount of beta-alanine ingested is decreased in0.4 g increments.
 42. The method of claim 32, wherein the amount ofbeta-alanine ingested is decreased in 0.5 g increments.
 43. The methodof claim 32, wherein the amount of beta-alanine ingested is decreased in0.6 g increments.
 44. The method of claim 32, wherein the amount ofbeta-alanine ingested is decreased in 0.7 g increments.
 45. The methodof claim 32, wherein the amount of beta-alanine ingested is decreased in0.8 g increments.
 46. The method of claim 32, wherein the amount ofbeta-alanine ingested is decreased in 0.9 g increments.
 47. The methodof claim 32, wherein the amount of beta-alanine ingested is decreased in1.0 g increments.
 48. The method of claim 32, wherein the amount ofbeta-alanine ingested is decreased to a minimum of 1.0 g.
 49. The methodof claim 32, wherein the decreasing the amount of beta-alanine ingestedin a 24 hour period for a period of time is from 3 days to 8 weeks. 50.The method of claim 49, where the decreasing the amount of beta-alanineingested in a 24 hour period for a period of time is from 5 days to 6weeks.
 51. The method of claim 50, wherein the decreasing the amount ofbeta-alanine ingested in a 24 hour period for a period of time is from 1week to 4 weeks.
 52. The method of claim 32, wherein the improvementfurther comprises increasing the amount of beta-alanine ingested in a 24hour period for a period of time following decreasing the amount ofbeta-alanine ingested in a 24 hour period.